1. Field of the Invention
This invention relates to special buffer solutions and their use for refolding polypeptides.
2. Description of Related Art
For commercial production of many polypeptides and proteins, recombinant DNA techniques have become the method of choice because of the large quantities that can be produced in bacteria and other host cells. Manufacturing recombinant protein involves transfecting or transforming host cells with DNA encoding the desired exogenous protein and growing the cells under conditions favoring expression of the recombinant protein. E. coli and yeast are favored as hosts because they can be made to produce recombinant proteins at high titers.
Numerous U.S. patents on general bacterial expression of recombinant-DNA-encoded proteins exist, including U.S. Pat. No. 4,565,785 on a recombinant DNA molecule comprising a bacterial gene for an extracellular or periplasmic carrier protein and non-bacterial gene; U.S. Pat. No. 4,673,641 on coproduction of a foreign polypeptide with an aggregate-forming polypeptide; U.S. Pat. No. 4,738,921 on an expression vector with a trp promoter/operator and trp LE fusion with a polypeptide such as insulin-like growth factor (IGF-I); U.S. Pat. No. 4,795,706 on expression control sequences to include with a foreign protein; and U.S. Pat. No. 4,710,473 on specific circular DNA plasmids such as those encoding IGF-I.
Under some conditions, certain heterologous proteins expressed in large quantities from bacterial hosts are precipitated within the cells in dense aggregates, recognized as bright spots visible within the enclosure of the cells under a phase-contrast microscope. These aggregates of precipitated proteins are referred to as "refractile bodies," and constitute a significant portion of the total cell protein. Brems et al., Biochemistry, 24: 7662 (1985). On the other hand, the aggregates of protein may not be visible under the phase contrast microscope, and the term "inclusion body" is often used to refer to the aggregates of protein whether visible or not under the phase-contrast microscope.
It has been found that the soluble proportion of high-level expressed protein in E. coli has been dramatically increased by lowering the temperature of fermentation to below 30.degree. C. A considerable fraction of various foreign proteins, i.e., human interferon-alpha (IFN-.alpha.2), interferon-gamma (IFN-.gamma.), and murine MX protein [Schein and Noteborn, Bio/Technology, 6: 291-294 (1988)] and human IFN-.beta. [Mizukami et al., Biotechnol. Lett., 8: 605-610 (1986)], stayed in solution. This procedure represents an alternative to renaturation of proteins recovered from refractile bodies, but requires an expression system that is efficiently induced at temperatures below 30.degree. C. The procedure is therefore not effective for all proteins.
For general review articles on refractile bodies, see Marston, supra; Mitraki and King, Bio/Technology, 7: 690 (1989); Marston and Hartley, Methods in Enzymol., 182: 264-276 (1990); Wetzel, "Protein Aggregation In Vivo: Bacterial Inclusion Bodies and Mammalian Amyloid," in Stability of Protein Pharmaceuticals: In Vivo Pathways of Degradation and Strategies for Protein Stabilization, Ahern and Manning (eds.) (Plenum Press, 1991); and Wetzel, "Enhanced Folding and Stabilization of Proteins by Suppression of Aggregation In Vitro and In Vivo," in Protein Engineering--A Practical Approach, Rees, A. R. et al. (eds.) (IRL Press at Oxford University Press, Oxford, 1991).
Recovery of the protein from these bodies has presented numerous problems, such as how to separate the protein encased within the cell from the cellular material and proteins harboring it, and how to recover the inclusion body protein in biologically active form. The recovered proteins are often predominantly biologically inactive because they are folded into a three-dimensional conformation different from that of active protein. For example, misfolded IGF-I with different disulfide bond pairs than found in native IGF-I has significantly reduced biological activity. Raschdorf et al., Biomedical and Environmental Mass Spectroscopy, 16: 3-8 (1988). Misfolding occurs either in the cell during fermentation or during the isolation procedure. Methods for refolding the proteins into the correct, biologically active conformation are essential for obtaining functional proteins.
Another property experienced by proteins during refolding is the tendency to produce disulfide-linked dimers, trimers, and multimers. Morris et al., Biochem. J., 268: 803-806 (1990); Toren et al., Anal. Biochem., 169: 287-299 (1988); Frank et al., in "Peptides: synthesis-structure-function,"ed. D. H. Rich and E. Gross, pp. 729-738 (Pierce Chemical Company: Rockford, Ill., 1981). This association phenomenon is very common during protein refolding, particularly at higher protein concentrations, and appears often to involve association through hydrophobic interaction of partially folded intermediates. Cleland and Wang, Biochemistry, 29: 11072-11078 (1990).
Protein folding is influenced by the nature of the medium containing the protein and by a combination of weak attractive or repellent intramolecular forces involved in hydrogen bonding, ionic bonding, and hydrophobic interactions. When pairs of cysteine residues are brought into close proximity as the peptide backbone folds, strong covalent disulfide bonds form between cysteine residues, serving to lock the tertiary conformation in place. Refolding protocols have been designed to break incorrect disulfide bonds, block random disulfide bonding, and allow refolding and correct disulfide bonding under conditions favorable to the formation of active conformer.
One series of techniques for recovering active protein from inclusion bodies involves solubilizing the inclusion bodies in strongly denaturing solutions and then optionally exchanging weakly denaturing solutions for the strongly denaturing solutions (or diluting the strongly denaturing solution), or using molecular sieve or high-speed centrifugation techniques. Such recovery methods, described, e.g., in U.S. Pat. Nos. 4,512,922; 4,518,256; 4,511,502; and 4,511,503, are regarded as being universally applicable, with only minor modifications, to the recovery of biologically active recombinant proteins from inclusion bodies. These methods seek to eliminate random disulfide bonding prior to coaxing the recombinant protein into its biologically active conformation through its other stabilizing forces.
In one method for recovering protein from inclusion bodies, the denatured protein desired to be refolded is further purified under reducing conditions that maintain the cysteine moieties of the protein as free sulfhydryl groups. The reducing agent is then diluted into an aqueous solution to enable the refolded protein to form the appropriate disulfide bonds in the presence of air or some other oxidizing agent. This enables refolding to be easily incorporated into the overall purification process.
In another approach, refolding of the recombinant protein takes place in the presence of both the reduced (R-S-E) and oxidized (R-S-S-R) forms of a sulfhydryl compound. This allows free sulfhydryl groups and disulfides to be formed and reformed constantly throughout the purification process. The reduced and oxidized forms of the sulfhydryl compound are provided in a buffer having sufficient denaturing power that all of the intermediate conformations of the protein remain soluble in the course of the unfolding and refolding. Urea is suggested as a suitable buffer medium.
The third alternative in this series is designed to break any disulfide bonds that may have formed incorrectly during isolation of the inclusion bodies and then to derivatize the available free sulfhydryl groups of the recombinant protein. This objective is achieved by sulfonating the protein to block random disulfide pairings, allowing the protein to refold correctly in a weakly denaturing solution, and then desulfonating the protein, under conditions that favor correct disulfide bonding. The desulfonation takes place in the presence of a sulfhydryl compound and a small amount of its corresponding oxidized form to ensure that suitable disulfide bonds will remain intact. The pH is raised to a value such that the sulfhydryl compound is at least partially in ionized form to enhance nucleophilic displacement of the sulfonate.
These refolding protocols, while practical for their universal utility, have not been shown necessarily to be maximally efficient with, for example, recombinant IGF-I.
The recovery of the biological activity requires a carefully monitored renaturation procedure and may be very difficult depending on the protein in question. A number of publications have appeared that report refolding attempts for individual proteins that are produced in bacterial hosts or are otherwise in a denatured or non-native form. For example, formation of a dimeric, biologically active macrophage-colony stimulating factor (M-CSF) after expression in E. coli is described in WO 88/8003 and by Halenbeck et al., Biotechnology, 7: 710-715 (1989). The procedures described involve the steps of initial solubilization of M-CSF monomers isolated from inclusion bodies under reducing conditions in a chaotropic environment comprising urea or guanidine hydrochloride, refolding achieved by stepwise dilution of the chaotropic agents, and final oxidation of the refolded molecules in the presence of air or a redox-system.
U.S. Pat. No. 4,923,967 and EP 361,830 describe a protocol for solubilizing and sulphitolysing refractile protein in denaturant, then exchanging solvent to precipitate the protein. The protein is resolubilized in denaturant and allowed to refold in the presence of reducing agent. The multiple steps required to achieve correct folding are time-consuming.
Methods for refolding proteins have been reported for several proteins such as interleukin-2 (IL-2) [Tsuji et al., Biochemistry, 26: 3129-3134 (1987); WO 88/8849 (which discloses on p. 17 use of high concentrations of copper as oxidant], growth hormone from various sources [George et al., DNA, 4: 273-281 (1984); Gill et al., Bio/Technology, 3: 643-646 (1985); Sekine et al., Proc. Natl. Acad. Sci. USA, 82: 4306-4310 (1985); U.S. Pat. No. 4,985,544, the lattermost reference involving adding a denaturing agent and reducing agent to solubilize the protein, removing the reducing agent, oxidizing the protein, and removing the denaturing agent], prochymosin [Green et al., J. Dairy Res., 52: 281-286 (1985)], urokinase [Winkler et al., Bio/Technology, 3: 990-1000 (1985)], somatotropin [U.S. Pat. No. 4,652,630, whereby urea is used for solubilization, and a mild oxidizing agent is then used for refolding], interferon-beta [EP 360,937 published Apr. 4, 1990], and tissue-plasminogen activator [Rudolph et al., in "623rd Biochem. Soc. Meeting," Canterbury (1987)]. See also Marston, Biochemical J., 240: 1-12 (1986). An additional folding procedure using the pro-sequence of the naturally occurring polypeptide to promote folding of a biologically inactive polypeptide to its active form, exemplified by subtilisin, is disclosed in U.S. Pat. No. 5,191,063.
In certain recovery techniques, up to at least 60% active foreign protein has been obtained. See, e.g., Boss et al., Nucl. Acids Res., 12: 3791-3806 (1984); Cabilly et al., Proc. Natl. Acad. Sci. USA, 81: 3273-3277 (1984); Marston et al., Bio/Technology, 2: 800-804 (1984); Rudolph et al., supra.
Additional representative literature on refolding of non-native proteins derived from different sources include a report that IL-2 and interferon-.beta. (IFN-.beta.) have been refolded using SDS for solubilization and Cu.sup.+2 ions as oxidation promoters of the fully reduced proteins. U.S. Pat. No. 4,572,798. A process for isolating recombinant refractile proteins as described in U.S. Pat. No. 4,620,948 involves using strongly denaturing solutions to solubilize the proteins, reducing conditions to facilitate correct folding, and denaturant replacement in the presence of air or other oxidizing agents to reform the disulfide bonds. The proteins to which the process can be applied include urokinase, human, bovine, and porcine growth hormone, interferon, tissue-type plasminogen activator, foot-and-mouth disease (FMD) coat protein, pro-renin, and a src protein.
A method for renaturing unfolded proteins including cytochrome c, ovalbumin, and trypsin inhibitor by reversibly binding the denatured protein to a solid matrix and stepwise renaturing it by diluting the denaturant is disclosed in WO 86/5809. A modified monomeric form of human platelet-derived growth factor (PDGF) expressed in E. coli has been S-sulfonated during purification to protect thiol moieties and then dimerized in the presence of oxidizing agents to yield the active protein. Hoppe et al., Biochemistry, 28: 2956-2960 (1989).
Additionally, EP 433,225 published Jun. 19, 1991 discloses a process for producing dimeric biologically active transforming growth factor-.beta. protein or a salt thereof wherein the denatured monomeric form of the protein is subjected to refolding conditions that include a solubilizing agent such as mild detergent, an organic, water-miscible solvent, and/or a phospholipid. U.S. Pat. No. 4,705,848 discloses the isolation of monomeric, biologically active growth hormone from inclusion bodies using one denaturing step with a guanidine salt and one renaturing step. See also Bowden et al., Bio/Technology, 9: 725-730 (1991) on .beta.-lactamase cytoplasmic and periplasmic inclusion bodies, and Samuelsson et al., Bio/Technology, 9: 731 (1991) on refolding of human interferon-gamma mutants. Moreover, Hejnaes et al., Protein Engineering, 5: 797-806 (1992) describes use of a chaotropic agent with IGF-I.
Several literature references exist on the production of IGF-I in bacteria. These include EP 128,733 published Dec. 19, 1984 and EP 135,094 published Mar. 27, 1985, which address expression of IGF-I in bacteria. EP 288,451 addresses use of lamB or ompF signal to secrete IGF-I in bacteria; Obukowicz et al., Mol. Gen. Genet., 215: 19-25 (1988) and Wong et al., Gene, 68: 193-203 (1988) teach similarly. EP 286,345 discloses fermentation of IGF-I using a lambda promoter.
In addition, methods have been suggested for preparing IGF-I as a fusion protein. For example, EP 130,166 discloses expression of fusion peptides in bacteria, and U.S. Pat. No. 5,019,500 and EP 219,814 disclose a fusion of IGF-I with a protective polypeptide for expression in bacteria. EP 264,074 discloses a two-cistronic met-IGF-I expression vector with a protective peptide of 500-50,000 molecular weight [see also U.S. Pat. No. 5,028,531 and Saito et al., J. Biochem., 101: 1281-1288 (1987)]. Other IGF-I fusion techniques include fusion with protective peptide from which a rop gene is cut off [EP 219,814], IGF-I multimer expression [Schulz et al., J. Bacteriol., 169: 5385-5392 (1987)], fusion of IGF-I with luteinizing hormone (LH) through a chemically clearable methionyl or tryptophan residue at the linking site [Saito et al., J. Biochem., 101: 123-134 (1987)], and fusion with superoxide dismutase. EP 196,056. Niwa et al., Ann. NY Acad. Sci., 469: 31-52 (1986) discusses the chemical synthesis, cloning, and successful expression of genes for IGF-I fused to another polypeptide. These methods utilizing fusion proteins, however, generally require a relatively long leader sequence and are directed to improving expression of the inclusion body protein, not to improving refolding of the denatured recombinant protein.
U.S. Pat. No. 5,158,875 describes a method for refolding recombinant IGF-I that involves cloning the IGF-I gene with a positively charged leader sequence prior to transfecting the DNA into the host cell. The additional positive charge on the amino terminus of the recombinant IGF-I promotes correct refolding when the solubilized protein is stirred for 2-16 hours in denaturant solution. Following refolding, the leader sequence is cleaved and the active recombinant protein is purified. However, this multistep process is burdensome, requiring additional materials and effort to clone a heterologous leader sequence in front of the IGF-I gene and then to remove the leader sequence from the purified protein.
Another method for facilitating in vitro refolding of recombinant IGF-I involves using a solubilized affinity fusion partner consisting of two IgG-binding domains (ZZ) derived from staphylococcal protein A. See Samuelsson et al., supra. This method uses the protein A domain as a solubilizer of misfolded and multimeric IGF-I. While this method does not use denaturing agents or redox chemicals, it involves the extra steps of fusing onto the IGF-I gene a separate gene and removing the polypeptide encoded by that gene after expression of the fusion gene.
Other investigators have described studies of IGF-I refolding involving disulfide exchange equilibration of refolding intermediates. For example, the refolding of IGF-I using redox buffers was investigated and the partially oxidized IGF-I forms produced were characterized by Hober et al., Biochemistry, 31: 1749-1756 (1992).
Disulfide exchange can also be modulated using the additive agent of peptidyl disulfide isomerase (PDI) or peptidyl prolyl isomerase (PPI). See, for example, JP Pat. Appln. No. 63294796 published Dec. 1, 1988; EP 413,440 published Feb. 20, 1991; and EP 293,793 published Dec. 7, 1988.
Enhancement of selected disulfide pairings by adding 50% methanol to buffer at low ionic strength has been reported by Snyder, J. Biol. Chem., 259: 7468-7472 (1984). The strategy involves enhancing formation of specific disulfide bonds by adjusting electrostatic factors in the medium to favor the juxtaposition of oppositely charged amino acids that border the selected cysteine residues. See also Tamura et al., abstract and poster presented at the Eleventh American Peptide Symposium on Jul. 11, 1989 advocating addition of acetonitrile, DMSO, methanol, or ethanol to improve the production of the correctly folded IGF-I.
A method for folding AlaGlu-IGF-I involving changing the redox potential by dialysis against a buffer containing from 20-40% v/v ethanol over a period of up to five hours and acidifying the mixture is disclosed in WO 92/03477 published Mar. 5, 1992.
Methanol was used at certain concentrations in the denaturation of ribonuclease. Lustig and Fink, Biochim. Biophys. Acta, 1119: 205-210 (1992). Studies by other laboratories indicate that moderate concentrations of alcohol can reduce association of insulin-like peptides under conditions that promote structure destabilization. Bryant et al., Biochemistry, 31: 5692-5698 (1992); Hua and Weiss, Biochim. Biophys. Acta, 1078: 101-110 (1991); Brems et al., Biochemistry, 29: 9289-9293 (1990); Ueda et al., JP 62-190199 published Jul. 20, 1987.
Research by other investigators has shown that solution polarity influences the propensity of peptides to acquire certain secondary structure. Jackson and Mantsch, Biochim Biophys. Acta, 1118: 139-143 (1992); Shibata et al., Biochemistry, 31: 5728-5733 (1992); Zhong and Johnson, Proc. Natl. Acad. Sci. USA, 89: 4462-4465 (1992). In general, reduced solution polarity appears to favor formation of alpha helix in short peptides. Jackson and Mantsch, supra. Spectroscopic studies on insulin also indicate that moderate concentrations of alcohols enhance alpha helix content. Hua and Weiss, supra.
There is a need for an efficient and inexpensive procedure for refolding polypeptides, including insoluble, misfolded IGF-I and others, into the correct conformation so that the biological activity of the polypeptide can be restored.
Accordingly, it is an object of the present invention to provide an efficient refolding method for polypeptides.
It is another object to provide a refolding method that does not utilize expensive disulfide-exchange reagents such as glutathione.
It is a further object to provide a refolding method that does not produce a product containing disulfide adducts.
It is a still further object to provide refolding conditions that are maximally repeatable, robust and scalable.
These and other objects will be apparent to those of ordinary skill in the art.